The objective of the proposed research is to identify and ascertain the function of novel genes involved in heart development. Towards this goal I have developed an in vitro cell culture system that will allow me to efficiently identify, mutagenize, and clone genes that are expressed in embryonic heart. Briefly, embryonic stem (ES) cells are transfected with a gene trap vector and are allowed to differentiate into cardiocytes in vitro. The gene trap randomly inserts into the coding region of genes, thereby disrupting their function and allowing for expression of a lacZ reporter in cells that express the disrupted gene. Differentiated heart cells derived from transfected ES cells are screened for expression of lacZ, as an indicator that a given ES cell clone harbors a disrupted cardiac gene. cDNAs of trapped genes expressed in heart will be generated with lacZ-specific primers, cloned, and subjected to rigorous molecular characterization. Preliminary results from this screen have yielded an ES cell clone that harbors a disrupted novel gene that is expressed in embryonic and neonatal myocardium. These cells will be injected into host blastocysts in order to make germline chimeras. The normal gene function of this gene may be inferred from morphological defects of transgenic embryos homozygous for the gene trap allele.